1. Field of the Invention
The present invention is related to the field of the detection of micro-organisms in the growth phase.
The present invention will find its application mainly in the field of industrial microbiology, for example in the pharmaceutical, biotechnological or agro-food industries.
The invention relates in particular to a device permitting an early detection of the forming of colonies, based on micro-organisms present in the sample, at the surface of a membrane or a solid or semi-solid growth medium.
2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98
Many techniques are presently implemented to permit the detection of contaminants, for example bacteria, in a sample to be tested.
The most common and oldest method consists of a deposition at the surface of an agar growth medium, where the latter can be more or less selective for one or several types of microorganisms. The medium is then incubated at the proper temperature for the growth of the micro-organism looked for, during a time period that may often extend up to several days.
Such a method has the drawback of requiring a relatively long incubation period in order to permit detection visible to the naked eye of the colonies that formed on the agar.
Also known from the state of the art is to perform a chain polymerization reaction, also referred to as PCR amplification, in order to determine the presence of specific micro-organisms within a sample, through amplification of a DNA or RNA sequence.
These methods have the drawback of requiring several DNA strands, i.e. several contaminating micro-organisms, generally at least several dozens of micro-organisms. Such methods are thus less sensitive than the methods based on the growth.
Also known is the possibility of using techniques consisting in marking the micro-organisms so as to emphasize the contrast between the light emitted by the micro-organisms and that emitted by the growth support. The use of specific fluorescent markers, the fluorophores, or of enzymes permitting to reveal the bioluminescence emitted for example by the [Alpha][Tau][Rho] (adenosine triphosphate), permits to detect the micro-organisms early, thanks to the use of optical systems sensitive to the characteristics of the emitted light, for example the wavelength or the intensity.
Known is thus namely the American patent application US 2003/0155528, which describes a method for detecting microorganisms, in which the latter are marked by means of appropriate fluorescent reagents permitting, on the one hand, in order to determine the quantity of micro-organisms and, on the other hand, to judge whether they are viable or dead cells.
These techniques can however prove heavy to be implemented and require the use of often expensive reagents and the presence of qualified labor. In addition, they are not well-suited for the detection of contaminants on a large number of samples, the marking operation being often difficult to be automated. Finally, these techniques exhibit a risk of contaminating the sample. Indeed, the addition of reagents requires a bringing into contact of said reagent with the micro-organisms to be detected.
In the state of the art are also known methods based on a use of the properties of light emitted naturally by the microorganisms, by detecting for example the self-fluorescence of said micro-organisms. Thus, it is possible to facilitate the distinction of colonies by using the contrast existing between the natural fluorescence emitted by said micro-organisms and the non-fluorescent support on which they have been arranged.
A method using this principle is described namely in international patent application WO 03/022999, in which determined optical properties of the colonies are used, such as the self-fluorescence.
These techniques permit indeed to facilitate the detection of colonies or of the self-fluorescent micro-organisms, the latter then presenting a better contrast with the membrane or the growth medium. However, the level of fluorescence generated naturally is of low amplitude, which does not permit to obtain fast detection times, compared for example with a marking with a specific fluorophore. In addition, the parasitic emission of natural fluorescence by the growth medium or by other particles present in the environment, such as dust, fibers of membranes or plastic particles proceeding from the support, can generate a falsely positive result.
Finally, techniques using systems with high optical magnification can be used to view colonies at early stages of development: this is the case for example for microscopes. Nevertheless, these devices are limited to detection on small surfaces, generally smaller than 1 mm<2>. Thus, the implementation of this type of techniques for the detection on one or several detection supports, such as membranes or agar media, proves both long, in the range of several minutes per cm<2>, and expensive, because of the necessity of using scanning systems.